siRNA下调20S蛋白酶体β5亚基抑制立氏立克次体细胞内增殖

Down-regulation of proteasome 20S subunit beta 5 inhibits the intracellular replication of Rickettsia rickettsii in host cells

摘要:

【目的】探究宿主20S蛋白酶体β5亚基（proteasome 20S subunit beta 5，PSMB5）对革兰氏阴性专性胞内寄生菌立氏立克次体胞内生长繁殖的影响。【方法】利用小干扰RNA转染Vero和THP-1宿主细胞，设置未转染细胞为空白对照组、无义小干扰RNA转染组为阴性对照组、PSMB5特异性小干扰RNA转染组为实验组，采用SYBR定量PCR和蛋白印迹法评价宿主细胞PSMB5变化水平；随后利用立氏立克次体以感染复数为0.1感染转染后细胞系，采用光镜观察、荧光显微镜观察和PCR定量方法检测细菌胞内繁殖变化水平。【结果】与si-NC阴性对照和空白对照相比，si-PSMB5能够显著降低宿主细胞PSMB5 mRNA转录水平和蛋白表水平；在随后立氏立克次体感染过程中，能够降低立氏立克次体的胞内细菌载量。【结论】下调宿主PSMB5基因表达能够抑制立氏立克次体胞内生长繁殖。

关键词:小干扰RNA;立氏立克次体;PSMB5;蛋白酶体;感染免疫

Abstract:

[Objective] This paper aims to explore the influence of host proteasome 20S subunit beta 5 (PSMB5) on the intracellular growth and reproduction of the Gram-negative obligate intracellular parasite Rickettsia rickettsii.[Methods] We transfected Vero and THP-1 cells with PSMB5-specific small interfering RNA (siRNA) (si-PSMB5 group), non-specific siRNA (negative control group), and solution (Mock group), respectively. Then, we verified the down-regulation of PSMB5 by RT-qPCR and Western blotting assay. Subsequently, we infected the transfected cells with R. rickettsii (multiplicity of infection=0.1) and detected R. rickettsii proliferation by light microscopy, indirect immunofluorescence staining, and RT-qPCR.[Results] Compared with the negative control and Mock control, si-PSMB5 significantly down-regulated the mRNA and protein levels of PSMB5 in host cells and R. rickettsii load in si-PSMB5-transfected cells was significantly reduced after R. rickettsii infection.[Conclusion] Down-regulation of host PSMB5 gene inhibits the intracellular growth of R. rickettsii.

Key words:small interfering RNA;Rickettsia rickettsii;PSMB5;proteasome;infection immunity